Microbiological information system

ABSTRACT

The present invention relates to an apparatus and a method for the classification of microorganisms. In particular, the invention relates to an apparatus and method for the classification of microorganisms with annotation of clinically and/or industrially relevant information. An apparatus according to the invention comprises a measuring apparatus for classifying a microorganism according to distinctive features, a database containing clinically and/or industrially relevant information of a plurality of microorganisms, and a calculation unit for comparing the results from the measuring apparatus to the information in the database for the annotation of the clinically and/or industrially relevant information to the microorganism, and in which the measuring apparatus, the database and the data analysis apparatus are provided with input and output means.

The invention relates to an apparatus for the classification ofmicroorganisms. In particular, the invention relates to an apparatus forthe classification of microorganisms with annotation of clinicallyand/or industrially relevant information.

Antibiotic resistance of infectious microorganisms is an importantproblem in microbiology, medicine and health care. Antibiotic resistancehas its origin in the evolution of microorganisms and the changes andspread of genetic material in and among microorganisms. The resistanceis strongly affected by the use of antibiotics and spreads inter alia inhospitals and through our food (Davies, 1997).

In many cases, plasmids and transposons are the vehicle for transfer ofgenetic information, such as complete genes, among microorganisms. Inthis manner, genes for, for instance, antibiotic resistance of anorganism can be transferred to another—not necessarily taxonomicallyrelated—organism.

Currently, many antibiotic resistance genes are known. For instance, itis known that resistance to the β-lactam penicillin can be encoded byinter alia pbp genes (penicillin binding protein) and by the bla operon(beta-lactamase). The genes responsible for antibiotic resistance arechromosome or plasmid-localized.

In many species of microorganisms, resistant strains are currentlyknown, including strains from the genera Streptococcus, Staphylococcus,Campylobacter, Haemophilus and Mycobacterium. In fact, allmicroorganisms are capable of producing antibiotic-resistant strains.

For health care, especially those microorganisms are of interest thathave an infectious nature, that is, they cause an infection in the humanbody which may or may not be contagious. It is particularly theseclinically relevant microorganisms that increasingly show resistance toexisting antimicrobial agents. A good example of this is MRSA, themeticillin-resistant form of the Staphylococcus aureus bacterium.

In detection and identification research of microorganisms and fordetermining an antibiotic susceptibility pattern, routine methods areused in the clinic. For the detection of a microorganism, generally aculture is started of blood, urine or tissue samples of the patient inquestion, it being observed whether metabolic activity of amicroorganism occurs therein.

When the culture is “positive”, next the antibiotic susceptibility ofthe microorganism can be tested, for instance by means of a so-calleddisk-diffusion test.

To determine the identity of the microorganism, in many cases, isolatedcolonies need to be obtained; for this purpose, a positive blood culturecan be plated on various nutrient media, after which the identity of themicroorganism is determined using biochemical tests (see: NCCLS,National Committee for Clinical Laboratory Standards. Approved standardM7-A. Methods for dilution antimicrobial susceptibility tests forbacteria that grow aerobically. National Committee for ClinicalLaboratory Standards, Villanova, Pa. 1985).

The methods above take up such an amount of time that most patients, incase of suspected infection, are administered a broad-spectrumantibiotic, that is effective against most known infectious diseases.However, the use of such agents has the disadvantage of inhibiting thegrowth of substantially all species of microorganisms, including thosein the intestinal flora of the patient which are necessary for a gooddigestion and a good barrier effect against intruding pathogens, withall its additional consequences. Another important disadvantage of abroad-spectrum antibiotic is that the corresponding resistance to theagent develops faster in the population of microorganisms.

For this reason, it is essential that the identity and the antibioticsusceptibility of an infectious microorganism be known as soon aspossible to the doctor in attendance, so that a specifically effectiveantibiotic can be administered.

On the basis of nucleic acid (DNA or RNA) detection techniques, in thepast decades, many methods have been developed for the detection andidentification of a microorganism, inter alia in a culture of a bloodsample. Such methods are aimed at, for instance, detecting a ribosomalRNA gene, or another specific genetic code by which a microorganism canbe recognized and identify a microorganism. In this connection, forinstance, a nucleic acid amplification reaction, such as a PCR reaction(Mullis, 1987; U.S. Pat. No. 4,683,202) or a NASBA reaction (Compton, J.1991; WO 91/02818) can be used, often in combination with, for instance,fluorogenic nucleic acid probes to detect the amplified nucleic acid.

In a further improvement of these genetic methods, methods have beendeveloped by which a plurality of specific genes or genetic codes can bedetermined in one single procedure or reaction, such as, for instance,in a so-called multiplex PCR reaction, making it possible to determine,in addition to the identity, the presence of specific antibioticresistance genes (see inter alia Maes et al., 2002).

Also, methods are currently known in which oligonucleotide chips ormicroarrays are used for the simultaneous identification of themicroorganism and the detection of antibiotic resistance genes (seeinter alia WO 98/20157 and WO 01/92573).

The use of oligonucleotide microarrays makes it possible for largeamounts of genetic information to be collected in a very short time. Itis inter alia known that DNA microarrays can be used to determine thecomplete nucleotide sequence of inter alia the rpoB gene, which, ifmutated, can cause resistance to rifampicin in mycobacteria.

As discussed above, in the clinic, antibiotic susceptibility andresistance are substantially determined by means of a phenotypic methodi.e. by determining a MIC value (minimal inhibiting concentration) usingthe disk-diffusion method. Antibiotic resistance, on the other hand, canalso be tested using a genetic method.

However, to be able to effectively treat infections, for clinicalpractice, it is inevitable to carry out conventional antibioticsusceptibility determinations based on determination of growthinhibition under the influence of various antibiotics. The fact is, onlysuch tests indicate whether specific treatment methods for reducing theinfection will be successful.

Knowledge concerning the presence of one or more resistance genes doesnot provide the doctor in attendance with information on the agent thatis most suitable to suppress and cure an infection in a patient. Also,the genetic method does not provide any quantitative information, suchas the degree of insusceptibility.

The DNA techniques currently available do not yet sufficiently meet theneed for fast, reproducible identification and the determination ofrelevant phenotypic characteristics of microorganisms, which is presentin laboratory diagnostics and which should be the basis for formulatingeffective therapy.

The genetic methods currently available for classification ofmicroorganisms practically give a doctor too little information for himto be able to effectively treat a patient.

In particular, there is a lack of insight into relevant clinical data ofboth microorganism and patient, such as co-medication, age and/or statusof the patient or other clinical empirical facts that may be related tothe qualitative outcome of the therapy, such as pathology of the strainin question and previous successful and unsuccessful treatment methods.

The present invention provides an apparatus for the classification ofmicroorganism with annotation of clinically relevant information.

An apparatus for classification of a microorganism with annotation ofclinically and/or industrially relevant information is capable ofmeeting the needs referred to above.

An apparatus according to the invention comprises a measuring apparatuswith a data file for classifying a microorganism according todistinctive features and a database with clinically and/or industriallyrelevant information of a plurality of microorganisms, with themeasuring apparatus and the database being provided with input andoutput means and being mutually coupled for the automatic annotation ofclinically and/or industrially relevant information to the classifiedmicroorganism.

The present invention further provides a method for classifying amicroorganism with annotation of clinically and/or industrially relevantinformation, comprising the use of an apparatus according to theinvention.

An apparatus and/or method according to the invention finds highlysuitable application in medical practice, such as diagnostic practiceaimed at the identification and antibiotic susceptibility determinationof clinically relevant microorganisms, prognostic practice aimed atpredicting the course of the disease or disorder, and therapeuticpractice, aimed at the formulation of an effective treatment plan by adoctor.

Further, an apparatus and/or method according to the invention findshighly suitable application utility in the field of process inspectionand process control of industrial microbiological processes, such asfood preparation processes and (microbiological) food safety research.

In the present invention, microorganisms are understood to be parasites,fungi, yeasts, bacteria and viruses of various taxonomical levels, suchas of different superkingdoms, kingdoms, phyla, classes, orders,families, genera, species or subspecies.

Clinically relevant microorganisms are generally understood to beorganisms that can cause an infection in the human body which may or maynot be contagious or transmittable. An apparatus and method according tothe invention are also suitable for the characterization of viruses inorder to be able to prescribe a suitable immunization procedure oranti-viral therapy.

Relevant microorganisms for the food industry are understood to be thosemicroorganisms that originally occur in the required ingredients and/orthe food product, or that are capable of surviving the productionprocess and/or the conservation process, and can thus cause a hazard orrisk to humans and animals.

An extensive list of clinically relevant microorganisms, which should byno means be construed as limiting, is shown in Table 1, with theorganisms being ordered according to taxonomical affiliation. TABLE 1Clinically relevant microorganisms Viruses Bacteria Superkingdom   CFBgroup     Bacteroides (inter alia B. fragilis)     Flavobacterium (interalia F. meningoseplicum)     Prevotella (inter alia P. intermedia)    Capnocytophaga (inter alia C. canimorsus)   Chlamydiales order    Chlamydia (inter alia C. trachomalis,     C. pneumoniae and C.psittaci)   Fusobacteria     Fusobacterium necrophorum    Streptobacillus moniliformis   Spirochaetales order     Borrelia(inter alia B. burgdorferi and     B. recurrentis)     Treponema (interalia T. pallidum)     Leptospira (inter alia L. interrogans)  Firmicutesphylum (Gram positive group)    Bifidobacteriales order     Gardnerella(inter alia G. vaginalis)    Lactobacillales order     Streptococcus(inter alia S. pneumoniae,     α-, β- and γ-hemolytic     and viridansand S. pyogenes)     Enterococcus (inter alia E. faecium and     E.faecalis)     Aerococcus (inter alia A. viridans)     Pediococcus (interalia P. acidilactici)     Leuconostoc (inter alia L.pseudomesenteroides)    Actinomycetales order     Mycobacterium (interalia M. tuberculosis,     M. lepra, M. africanum,     M. bovis and M.avium)     Nocardia (inter alia N. asteroides)     Corynebacterium(inter alia C. diphtheriae)     Micrococcus (inter alia M. luteus)    Actinomyces (inter alia A. israelii)     Propionibacteriumpropionicum     Brevibacterium (inter alia B. linens)    Mycoplasmatalesorder     Mycoplasma (inter alia M. pneumoniae)    Bacillales order    Staphylococcus (inter alia S. aureus     and S. pyogenes)    Alicyclobacillus (inter alia     Alicyclobacillus acidocaldarius)    Listeria (inter alia L. monocytogenes)     Bacillus (inter alia B.anthracis)     Gemella (inter alia G. morbillorum)    Clostridialesorder     Clostridium (inter alia C. botulinum, C. diffilie,     C.perfringens and C. tetani)     Peptostreptococcus (inter alia P.prevotii)     Veillonella (inter alia V. parvula)  Proteobacteria phylum  Alpha subdivision Class    Rickettsiales order     Rickettsiaceaefamily      Rickettsia (inter alia R. prowazekii      and R. typhi)     Ehrlichia (inter alia E canis, E. chaffeensis      and E.phagocytophila)      Cowdria (inter alia C. ruminantium)     Neorickellsia (inter alia N. helminthoeca)      Anaplasma (interalia A. marginale      and A. ovis)      Wolbachia (inter alia W.pipientis)    Rhizobacteriaceae group     Brucellaceae family     Brucella (inter alia B. melitensis      biovar abortus and B. m.biovar      canis)     Bartonellaceae family      Bartonella (inter aliaB. bacilliformis,      B. henselae and B. quintana)   Beta subdidvisionClass     Alcaligenaceae family      Alcaligenes (inter alia A.faecalis)      Bordetella (inter alia B. pertussis)     Neisseriaceaefamily      Neisseria (inter alia N. meningitidis      and N.gonorrhoeae)      Kingella (inter alia K. denitrificans)      Eikenella(inter alia E. corrodens)      Chromobacterium (o., a. C. violaceum)    Burkholderia group      Burkholderia (inter alia B. cepacia)   Gammasubdivision Class     Aeromonadaceae family      Aeromonas    Moraxellaceae family      Acinetobacter (inter alia A. lwoffii and     A. baumannii)      Moraxella (inter alia M. catarrhalis)    Enterobacteriaceae family      Escherichiae (inter alia E. coli)     Klebsiellae (inter alia K. pneumoniae)      Salmonellae (inter aliaS. typhimurium      and S. enteritidis)      Shigella (inter alia S.dysenteriac)      Edwardsiella (inter alia E. tarda)      Yersinia(inter alia Y. pestis)      Citrobacter (inter alia C. freundii)     Proteus (inter alia P. mirabilis)      Morganella morganii     Providencia (inter alia P. alcalifaciens)      Serratia (inter aliaS. marcescens)      Plesiomonas (inter alia P. shigelloides)    Legionellaceae family/Coxiella group      Legionella (inter alia L.pneumophila      and L. micdadei)      Coxiella (inter alia C. burnetii)     Rickettsiella (inter alia R. popilliae)      Tatlockia (inter aliaT. micdadei)      Fluoribacter (inter alia F. dumoffii)    Pasteurellaceae family      Haemophilus (inter alia H. influenzae     and H. ducreyi)      Pasteurella (inter alia P. multocida)    Pseudomonadaceae family      Pseudomonos (inter alia P. aeruginosa     and P. cepacia)     Francisella group      Francisella (inter aliaF. tularensis)     Vibrionaceas family      Vibrio (inter alia V.cholerae, V. vulnificus      and V. parahaemolyticus)     Xanthomonasgroup      Stenotrophomonas (inter alia S. mallophila)   Epsilonsubdivision Class     Campylobacter group      Campylobacter (inter aliaC. jejuni)      Helicobacter (inter alia H. pylari, H. cinaedi      andH. fennelliae) Eukaryota Superkingdom  Protista Kingdom   Trichomonadidaorder    Trichomonas (inter alia T. vaginalis)   Microsporida order   Enterocytozoon bieneusi   Amoebida order    Acanthamoeba (inter aliaA. castellani)    Entamoeba (inter alia E. histolytica)   Eucoccidiidaorder      Cryptosporidium (inter alia C. parvum)   Diplomonadidae order     Giardia lamblia   Eimeriida order      Cryptosporidium (inter aliaC. parvum)      Taxoplasma gondii      Neospora caninum   Haemosporidaorder      Plasmodium (inter alia P. falciparum)   Kinetoplaslida order     Trypanosoma (inter alia T. brucei)      Leishmania donovani  FungiKingdom      Acremonium      Aspergillus (inter alia A. fumigatus)     Beauveria      Fusarium      Histoplasma (inter alia H. duboisii)     Paecilomyces      Penicillium      Scopulariopsis      Trichophyton(inter alia T. rubrum and      T. mentagrophytes)      Cryptococcus(inter alia C. neoformans)      Coccidioides (inter alia C. immitis)     Candida (inter alia C. albicans)      Blastomyces      Malassezia     Pneumocystosis (inter alia P. carinii) Viruses  DNA viruses  Herpesviridae Herpes simplex virus type 1 Varicella zoster virusEpstein Barr virus Human cytomegalovirus Human herpesvirus 6  Adenoviridae Human adenoviruses   Papovaviridae Human papillamaviruses  Hepadnaviridae Hepatitis B virus   Poxviridae Vaccinia virus  Parvoviridae B19 parvovirus  RNA viruses   Picornaviridae PoliovirusesEchoviruses Coxsackieviruses Hepatitis A virus Human rhinoviruses  Caliciviridae Norwalk virus   Paramyxoviridae Parainfluenza virusesMeasles virus Respiratory syncytial virus   Orthomyxoviridae Influenxavirus   Rhabdoviridae Rabies virus   Filoviridae Ebola and Marburgviruses   Retroviridae Human immunodeficiency virus type-1 and -2  Togaviridae Rubella virus   Flaviviridae Yellow fever virus Denguevirus   Reoviridae Human rotaviruses   Bunyaviridae Pulmonary SyndromeHantavirus Hantaan virus   Arenaviridae Lassa virus   CoronaviridaeHuman coronaviruses   Astroviridae Human astrovirusesKarolinska Instituted Library Bacterial Infections and Mycoses(www.mic.ki.se); Atlas of Medical Parasitology, Carlo Denegri Foundation(www.cdfound.to.it); NCBI taxonomy database (www.ncbi.nlm.nih.gov);University of Rochester Medical Center Dept. of Microbiology andImmunology (www.urmc.rochester.edu)

A measuring apparatus for classifying a microorganism according todistinctive features according to the invention can comprise a single ormultiple measuring apparatus.

For classifying an organism, preferably a measuring apparatus is used inthe invention which can be used to measure morphologically,physiologically, serologically, pathologically, taxonomically and/orgenetically distinctive features between microorganisms.

In the context of the present invention, a morphological feature of amicroorganism is understood to refer to an externally observable featuresuch as the form of the organism; the possession of a specificbiochemical substance, for instance a membrane peptide, a pigment, a(glyco)protein, a lipid or a cell wall component such as mycolic acid;the possession or absence of a specific receptor; the production ofspores or cysts; the possession of flagella; growing in chains or infilaments, or another external feature such as a cell or colonymorphology; or a coloring characteristic.

In the context of the present invention, a physiological feature of amicroorganism is meant to refer to a specific catabolic characteristicsuch as proteolysis or a capability to grow on specific substrates suchas polysaccharides, proteins, fats or nucleic acids; specific nutrientrequirements; the possession of specific metabolic routes; a sensitivityto oxygen or a susceptibility to an antibiotic; a temperature or aciditydependence; the production of a specific metabolic final product; thesecretion of a bacteriocin or antibiotic; the production of a gas; themanner of energy supply of the organism; the size, composition oranother feature of the collection of proteins in the cell (theproteome); or a feature of the collection of low-molecular organicsubstances in the cell (the metabolome).

In the context of the present invention, a serological feature is meantto refer to the capability to react with a specific antibody ormonoclonal; the possession or absence of specific surface antigens orepitopes such as glycolipids or glycoproteins.

In the context of the present invention, a pathological feature of amicroorganism is meant to refer to a capability to infect cells; toxinsecretion; a manner in which an infection progresses; a hemolyticcharacteristic or other pathological feature, such as the naturalhabitat or the tissue or cell type that is affected by the organism.

In the context of the present invention, a taxonomical feature isdefined as a phenotypic feature, such as a morphological feature, aphysiological feature, a serological or pathological feature asdescribed above, on the basis of which a microorganism is usuallytaxonomically identified, but can also comprise a genetic feature on thebasis of which the phylogenetic lineage can be determined, and on thebasis of which a microorganism can also be taxonomically identified.

In the context of the present invention, a genetic feature is meant torefer to a specific chromosomal or extra-chromosomal distinctivenucleotide sequence of a nucleic acid such as a DNA and/or an RNA; aspecific genetic code or a gene; a linear or circular chromosome; thesize or another feature of the genome; the G+C content; the presence ofplasmids; the possession of specific transposons, integrons or insertionsequences; the composition or size of the expression profile(transcriptome).

A measuring apparatus according to the invention for classifying amicroorganism according to distinctive features can be arranged suchthat it can measure different or the same types of distinctive featuresbetween microorganisms, such as morphological, physiological,serological, pathological, taxonomical and/or genetic features.

If different types of distinctive features between microorganisms can bemeasured using an apparatus according to the invention, these featurescan be measured sequentially and/or simultaneously.

A measuring apparatus according to the invention can inter alia be usedto determine a genetically distinctive feature of a microorganism. Sucha genetically distinctive feature can comprise, for instance, aresistance gene to which a specific antibiotic-resistant phenotype isrelated. This can be understood to refer to inter alia the mecA geneencoding the penicillin-binding protein 2a in Staphylococcus, makingthis bacterium insusceptible to substantially all β-lactam antibiotics,including meticillin.

Other resistance genes that can be measured using a measuring apparatusaccording to the invention that can measure a genetically distinctivefeature of a microorganism, are inter alia the aac(6′) gene in Serratiamarcescens or Klebsiella pneumoniae, which causes resistance toaminoglycosides, such as netilmicin and gentamicin, or resistance genessuch as nptII (kanamycin resistance), vanA, B and C (vancomaycinresistance), ermA, B and C msrA (macrolide resistance), gyrA, grlA(quinolone resistance), bla (β-lactam resistance), vat, vga(streptogramin resistance), or sul and int (sulfonamide resistance).

In addition to measuring specific resistance genes, a measuringapparatus according to the invention which can measure a geneticallydistinctive feature of a microorganism can also be used to measuremutations in specific genes. Mutations in the PfCRT transmembraneprotein of the digestive vacuole or in the PfMRD1 gene for theP-glycoprotein homolog 1 (Pgh1) of Plasmodium falciparum cause, forinstance, insusceptibility of this parasite to agents such aschloroquine and can be measured using a measuring apparatus according tothe invention.

In an alternative embodiment, mutations in, for instance, a ribosomalRNA gene can be detected, on the basis of which taxonomical orphylogenetic information and/or features can be determined.

Also, other genes or genetic codes, the function of which is yet isunknown, can provide relevant distinctive features betweenmicroorganisms which can be measured in an embodiment according to thepresent invention, as long as they represent a distinctive feature for aclassification of a microorganism according to the invention.

For measuring distinctive features in a microorganism using a measuringapparatus according to the invention, preferably markers are used in anapparatus according to the invention.

In the present invention, a marker is defined as a characteristicdistinctive feature of a microorganism that can be measured, preferablyby using molecular biological methods, for instance by determining ormeasuring the distinctive feature by using complementary bindingpartners, such as complementary nucleic acids or complementaryoligonucleotide probes in the case of a genetic feature, or an antibodyor monoclonal or another binding partner in the case of a physiologicalfeature such as a protein. Suitable markers or binding partners areassumed to be known to a skilled person. The detection of bindingbetween the complementary binding partner and the marker can befacilitated by using labels.

When in the present invention nucleic acid markers or genetic markersare referred to, this is understood to mean complementary bindingpartners as well.

Markers can comprise genetic or phenotypic markers. Preferably, in ameasuring apparatus according to the present invention, genetic and/orphysiological markers, such as protein markers, are used. With greatpreference, genetic markers are used.

In addition to the fact that genetic markers can be used in a measuringapparatus according to the present invention for determining thepresence of (known) genetic distinctive features in a microorganism,they can also be used highly suitably to determine phenotypic features,such as for instance antibiotic susceptibility and/or antibioticresistance.

To detect relevant genetic and/or phenotypic features, genetic markerscan be used highly suitably in an apparatus according to the presentinvention. Many genetic markers that are suitable for use in anapparatus according to the invention are known to a skilled person. Askilled person can also identify and produce suitable genetic markershimself in a simple manner.

Methods for identifying genetic markers related to specific phenotypiccharacteristics of microorganisms are known to a skilled person (seeinter alia WO 01/83813). For instance, so-called polymorphisms can bedetected using methods described in U.S. Pat. No. 6,300,063. For thispurpose, known genetic fingerprint methods can also be used (see Muellerand Wolfenbarger for an overview), such as AFLP (Vos et al., 1995),RAPD, or RFLP (Botstein et al., 1980) or techniques derived therefromsuch as Ribotyping.

In addition to the fact that such fingerprint methods can be used toidentify genetic markers, they can also result in a fingerprint whichcan be used to distinguish microorganisms. As such, these fingerprintmethods and the instruments and equipment available for these findsuitable application utility in an apparatus and method according to theinvention for classifying a microorganism according to distinctivefeatures.

In the present invention, genetic markers such as RFLP markers (see, forinstance, U.S. Pat. No. 5,324,631), RAPDs (see, for instance,Aufauvre-Brown et al., 1992), AFLP markers (see, for instance, EP0,534,858) SSR markers (see, for instance, U.S. Pat. No. 5,075,217) andSNP markers McEwen et al., 2000) can be used.

A method comprising the use of an apparatus according to the inventionin an embodiment in which genetic markers are used, preferably comprisesa step for determining the presence or absence of a specific distinctivefeature or a marker in a nucleic acid, such as a DNA or RNA, of amicroorganism, for instance by means of fingerprinting or using amicroarray, such as a DNA array, an oligonucleotide array or, in generalterms, a nucleic acid array.

In a preferred embodiment, the invention relates to an apparatus inwhich a nucleic acid array is used, which comprises oligonucleotides ornucleic acid sequences of genetic markers or complements thereofimmobilized on a carrier surface.

Genetic markers can also be used for the amplification of nucleic acidsequences which are associated to a specific distinctive feature, in thepresent case as, for instance, primers. Methods to characterize suchamplified products, such as electrophoresis, chromatography, sequencingor mass spectrometry, are known to a skilled person.

To improve hybridization characteristics of oligonucleotides or nucleicacid sequences, specific nucleic acid analogs can be used which canenter into a sequence-specific interaction equal to that of the naturalphosphodiester nucleic acid, such as phosphorothioate ormethylphosphonate oligonucleotides or peptide nucleic acid (PNA)oligonucleotides.

In a method according to the present invention, use of a DNA array ispreferred. Such arrays of oligonucleotides then comprise thecomplementary binding partners of genetic markers, and are also part ofthe present invention.

The production of an oligonucleotide array according to the inventioncan be carried out using methods known to a skilled person. Theproduction and the use of solid-carrier nucleic acid arrays for thedetection of specific nucleic acid sequences has frequently beendescribed (U.S. Pat. No. 5,571,639; Sapolsky et al., 1999, Genet.Anal.-Biomolecular Eng. 14, 187-192; Shena et al., 1995, Science 270,467-470; Sheldon et al., 1993, Clinical Chem. 39, 718-719; Fodor et al.,1991, Science 251, 767-773).

A skilled person will be capable of obtaining arrays by his own designand the corresponding array reading equipment from specialized suppliers(for instance Affymetrix Corp., Santa Clara, Calif., USA for DNA arraysand Ciphergen Biosystems, Fremont, Calif., USA for protein arrays).

A DNA array according to the present invention can comprise, forinstance, between 10 and 200,000 oligonucleotides which are specific forspecific sequences in the form of genetic markers. Also, an array cancomprise oligonucleotides which comprise genetic markers such as SNPsand microsatellite markers. Methods for designing sets ofoligonucleotide probes for simultaneous analysis of nucleic acids, suchas expression products of genes, are described in inter alia EP0,799,897.

It is advantageous when the melting point of the oligonucleotides issubstantially in the same range to enable hybridization in uniformconditions.

In particular, synthesis of the oligonucleotides can be carried outdirectly on the solid carrier surface of the array, such as for exampleusing a photochemical synthesis technique described in U.S. Pat. No.5,424,186 or using an ink-jet technique. In an alternative embodiment,the oligonucleotides can be synthesized ex situ and bound to the solidcarrier surface. In this case, it is advantageous if the carrier surfacehas been chemically modified prior to the application of theoligonucleotides to enable binding between the oligonucleotides and thecarrier surface, optionally using a hydrogel matrix or additionalorganic or inorganic linkers between the oligonucleotide and the carriersurface of the array. Addressing the various oligonucleotides on thesurface can be done electronically, mechanically or using an ink-jet.

The hybridization conditions will depend on the nucleic acid used assample material, but can be optimized in a simple manner using methodsknown to a skilled person. For this purpose, inter alia the saltcontent, the pH and the temperature of the hybridization can beadjusted. Optionally, methods can be used to electronically control thestringency of the hybridization, as known from U.S. Pat. No. 6,017,696.

The detection of the hybridization spots can be cared out using labelssuch as radioisotope labels or fluorescent labels, using field effectmeasurements, using optoelectrochemical methods, piezo-electric methods,or ellipsometry, measurement using optical fibers or mass spectrometry.Also, telemetry can be used to investigate the presence of markers inthe starting nucleic acid.

Prior to hybridization, the nucleic acid fragments can highly suitablybe labeled, for instance using a fluorescent label or a radioisotope oranother label, to facilitate the detection of these fragments hybridizedto the oligonucleotides on the array. Depending on the selected methodof detection, a skilled person will be able to use a suitable label.

A method comprising the use of an apparatus according to the inventionpreferably comprises a step for determining the presence or absence of aspecific distinctive feature in a microorganism, in which a samplecontaining material of the microorganism is brought into contact withthe measuring apparatus. Such a contact location can very suitably serveas input means for the measuring apparatus.

The apparatus according to the invention comprises a measuring apparatuswith a data file for classifying a microorganism according todistinctive features. Such a data file preferably comprises dataconcerning markers and distinctive features of microorganisms asdescribed above. The measuring apparatus will further comprise acalculation unit to process the measuring results obtained by using themeasuring apparatus.

In an embodiment of the method according to the invention in which anucleic acid array is used as contact location of a measuring apparatus,nucleic acid of a microorganism or fragments thereof are brought intocontact with the array of nucleic acid markers.

Methods for obtaining genetic information using nucleic acid arrays areknown in the literature (see inter alia Chee et al., 1996).

A measuring apparatus for measuring distinctive features in amicroorganism according to the invention can, in an alternativeembodiment, be formed by, for instance, an apparatus for “MatrixAssisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry”(MALDI-TOF-MS), “Surface-Enhanced Laser Desorption/IonizationTime-of-Flight Mass Spectrometry” (SELDI-TOF-MS), “High PerformanceLiquid Chromatography tandem Mass Spectrometry” (HPLC-MS/MS). “surfaceplasmon resonance” (SRP), optionally using markers.

A measuring apparatus according to the present invention can be arrangedfor single or plural measurements, but is preferably arranged for plural(simultaneous) measurements. A measuring apparatus according to theinvention is further preferably arranged for receiving, handling,processing, classifying, categorizing, filing, outputting and/or storingresults of measurements of distinctive features for classifying amicroorganism according to the invention and is, for this purpose,preferably provided with means such as a calculation unit and a datafile, which data file comprises distinctive features of microorganismsfor the classification of a microorganism.

A measuring apparatus with a data file of distinctive features ispreferably also arranged for filing measuring results obtained by usinga measuring apparatus according to the invention.

A calculation unit can be used as part of the measuring apparatus, butcan also be used externally or separate from the measuring apparatus. Acalculation unit is arranged for performing calculations, in whichresults obtained using a measuring apparatus according to the inventioncan be compared to distinctive features in a data file. For thispurpose, a calculation unit preferably comprises input and output meansthat can be connected to corresponding output means of a measuringapparatus and corresponding input and output means of a data file withdistinctive features.

Further, a calculation unit according to the invention is preferablyarranged for categorizing and classifying distinctive features ofmicroorganisms obtained using a measuring apparatus according to theinvention according to taxonomical position of a microorganism. For thispurpose, preferably, the results are sent as data from a measuringapparatus according to the invention to an input of a calculation unitaccording to the invention, to be subsequently categorized or classifiedby a calculation unit according to the invention.

A calculation unit according to the invention is preferably amathematical calculation unit for solving algorithmic comparisons inwhich categorized and classified distinctive features of microorganismsare inter alia statistically compared to relevant information from adatabase and in which a calculation result is outputted, preferably to adisplay. Preferably, a calculation unit according to the inventioncomprises a microprocessor unit.

With great preference, for comparison analyses by a calculation unit,algorithmic computer analysis methods are used such as self-organizingmaps, hierarchic clustering, multidimensional scaling, principalcomponent analysis, supervised learning, k-nearest neighbors, supportvector machines, discriminant analysis or partial least square methods.Such methods are known to a skilled person.

A calculation unit according to the invention is arranged forclassifying a microorganism according to distinctive features.Preferably, this classification comprises a taxonomical classification.With great preference, the classification results in a detailedtaxonomical positioning or identification of a microorganism.

An apparatus for classifying a microorganism with annotation ofclinically and/or industrially relevant information according to theinvention further comprises a database containing clinically and/orindustrially relevant information of a plurality of microorganisms andpreferably for a plurality of clinical and/or industrial situations.

A database containing clinically and/or industrially relevantinformation according to the invention comprises at least one memorylocation for storage of all possible types of information which are inany way related to microorganisms that play a role in a clinical and/orindustrial environment, preferably digitized. A database according tothe invention can comprise a combination of clinically relevantinformation and industrially relevant information. Preferably, adatabase according to the invention comprises relevant information forone of the two environments, with this information being limited to aspecific use of the apparatus according to the invention.

Information that is in any way related to microorganisms that play arole in a clinical environment is preferably categorized according todifferent taxonomical groups and based on different taxonomical levelsof microorganisms. Furthermore, information may be categorized accordingto different natural or artificial, known or uncharacterized mixedpopulations of microorganisms.

Such information can, for instance, comprise information concerning themicroorganisms themselves and the nature of the infection they cause,such as geographical origin of the microorganism, the incubation time inwhich disease symptoms become manifest after exposure, the antibioticsof which an effect on the microorganism is known, from informationconcerning patients who suffer or have suffered from the infection, suchas average age, the co-medication, the health status, the ethnic origin,the epidemiological origin, the family relationship, etc., and/or frominformation concerning treatment methods which have already been usedfor specific infections, such as medication, diet, relationfood/environment.

Information that is in any way related to microorganisms that play arole in an industrial environment can, for instance, compriseinformation concerning the microorganisms and the nature of the processthey carry out, such as information concerning physical and biologicalprocess parameters, such as pH, Aw (water activity) and the temperaturesensitivity and/or information concerning process treatments which havealready been used for specific industrial processes, such as cooling,freezing, pasteurizing, sterilizing, but also alternative techniquessuch as the use of high pressure, light, electric or magnetic fields andradiation. Also, this may involve effects resulting from the effects ofcleaning and the use of disinfectants.

A database according to the invention is provided with a means intendedfor data input and is preferably connected to a data presentation unitby means of an output.

It is possible to add new information to the database or to removeinformation therefrom, which yields a database with a dynamic character.By adding new relevant information, the size and the detail of thedatabase will increase, thus providing an increasingly better basis forresults.

A database containing clinically or industrially relevant informationaccording to the invention and a data file containing distinctivefeatures as part of a measuring apparatus for classifying amicroorganism according to the invention can be combined in analternative embodiment.

Annotation of clinically or industrially relevant information which ispresent in a database according to the invention is done by combiningthe information of the classification of the microorganism or of themicroorganisms obtained using the measuring apparatus according to theinvention with the relevant information from the database for themicroorganism in question or a higher taxonomical level thereof.

The output of an apparatus according to the invention can, for instance,take place in the form of a chance that a specific proposed therapy orprocess treatment method will be successful, or can, for instance, takeplace in the form of a proposal for a highly suitable therapy or processtreatment method.

References

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1. An apparatus for classification of a microorganism with annotation ofclinically and/or industrially relevant information, comprising ameasuring apparatus containing a calculation unit and a data file forclassifying a microorganism according to distinctive features and adatabase containing clinically and/or industrially relevant informationof a plurality of microorganisms, wherein said measuring apparatus andsaid database are provided with input and output means and are mutuallycoupled for the automatic annotation of said clinically and/orindustrially relevant information to the classified microorganism.
 2. Anapparatus according to claim 1, wherein said microorganisms compriseclinically relevant microorganisms.
 3. An apparatus according to claim1, wherein said measuring apparatus comprises an array of physiologicaland/or genetic markers.
 4. An apparatus according to claim 3, whereinsaid genetic markers comprise oligonucleotides.
 5. An apparatusaccording to claim 1, wherein the data file of said measuring apparatuscomprises morphological, physiological, serological, pathological,taxonomical or genetic features.
 6. An apparatus according to claim 1,wherein said database comprises clinically relevant information and/orindustrially relevant information.
 7. An apparatus according to claim 6,wherein said clinically relevant information comprises informationconcerning microorganisms, concerning the nature of an infection,concerning patients and/or concerning treatment methods.
 8. An apparatusaccording to claim 6, wherein said industrially relevant informationcomprises information concerning microorganisms, concerning the natureof a process, concerning process parameters and/or concerning processtreatments.
 9. An apparatus according to claim 1, wherein saidcalculation unit is arranged for performing algorithmic computeranalyses selected from the group consisting of self-organizing maps,hierarchic clustering, multidimensional scaling, principal componentanalysis, supervised learning, k-nearest neighbors, support vectormachines, discriminant analysis and partial least square.
 10. Anapparatus according to any claim 1, wherein said calculation unit isarranged for categorizing and classifying distinctive features ofmicroorganisms obtained by using a measuring apparatus according to theinvention.
 11. A method for classifying a microorganism with annotationof clinically and/or industrially relevant information, comprising theuse of an apparatus according to the invention.
 12. A method accordingto claim 11, wherein a sample containing material of a microorganism isbrought into contact with a measuring apparatus according to theinvention at a contact location intended for the purpose to measure thepresence or absence of a particular distinctive feature in amicroorganism.